锦鲤疱疹病毒ORF68部分基因原核表达及单克隆抗体的制备

发布时间：2018-06-04 14:12

本文选题：锦鲤疱疹病毒 + ORF68基因　；参考：《吉林农业大学》2017年硕士论文

【摘要】：KHVD给世界多个国家的鲤鱼及锦鲤养殖业造成了严重的经济损失,引起这种现象主要是由于没有快速安全有效的诊断方法,不能尽早发现及时诊断,应用单克隆抗体技术,可以在发病初期进行诊断尽早发现问题及时解决问题。单克隆抗体技术应用于锦鲤疱疹病毒病起步较晚,目前国外只有3篇相关报道,国内仍然没有成功案例,因此本研究致力于筛选出抗锦鲤疱疹病毒的单克隆抗体,推动鲤鱼的健康生长,对于我国鲤鱼养殖具有重大的意义。以本实验室分离保存的KHV-CJ株DNA为模板,采用PCR扩增技术,分别扩增出753 bp、897bp和1500 bp大小的ORF68基因片段,将三段基因插入到pMD18-T载体中,通过PCR和双酶切鉴定后,确定克隆质粒pMD18T-753、pMD18-T-807和pMD18-T-1500构建成功。经BLAST比对,获得的目的基因片段同源性可达到100%。分别用三段基因构建重组表达质粒,转入BL21表达菌中,经IPTG诱导后对重组蛋白进行可溶性分析表明,本实验重组蛋白可以在大肠杆菌包涵体中和可溶性形式同时存在,分子量分别为45.61kDa、47.59kDa和73kDa。采用带有His标记的Ni离子层析柱进行纯化,经测定重组蛋白浓度分别为:重组蛋白pET32a-753:0.46mg/ml,重组蛋白pET32a-807:0.57mg/ml,重组蛋白pET32a-1500:0.51mg/ml。按照免疫程序进行小鼠免疫,ELISA检测其免疫后的血清效价,将免疫小鼠脾细胞与SP2/0细胞融合,经HAT条件培养基筛选后,使用ELISA法检测培养基上清效价,初步筛选确定阳性克隆后进行多次筛选,根据OD450nm值,在其中筛选出表达量较高的细胞株,进行有限稀释筛选出的阳性细胞扩大培养后,注射小鼠腹腔,制备腹水。对制备的腹水进行反应原性、类别鉴定。结果表明:通过对单克隆细胞株培养基上清进行初次检测选择出227株阳性单克隆细胞株,进行多次检测后筛选出较稳定15株阳性结果,根据OD450nm值,在其中选出5株OD450nm值较高的,分别命名为:807-1D3、807-1D6、807-3E5、1500-3D2、1500-4G2,最终筛选出1株较稳定的阳性株:807-1D6,其抗体类别为IgG1,通过Western-blot确定其能识别KHV重组蛋白pET32a-807。
[Abstract]:KHVD has caused serious economic losses to carp and koi culture in many countries in the world. This phenomenon is mainly due to the lack of rapid, safe and effective diagnostic methods, the lack of prompt diagnosis as early as possible, and the application of monoclonal antibody technology. Can be diagnosed in the early stage of diagnosis as early as possible to solve the problem. The application of monoclonal antibody technology to koi herpesvirus disease started late. At present, there are only three related reports in foreign countries, but there are still no successful cases in China. Therefore, this study is devoted to screening monoclonal antibodies against koi herpesvirus. It is of great significance to promote the healthy growth of carp in China. Using the DNA of the KHV-CJ strain isolated and preserved in our laboratory as template, the ORF68 gene fragments of 753 BP, 897 BP and 1500 BP were amplified by PCR amplification technique. The three fragments were inserted into the pMD18-T vector and identified by PCR and double enzyme digestion. The cloned plasmid pMD18T-753pMD18-T-807 and pMD18-T-1500 were successfully constructed. The homology of the target gene fragment obtained by BLAST was 100%. The recombinant expression plasmids were constructed with three segments of genes and transferred into BL21 expression bacteria. The soluble analysis of the recombinant protein induced by IPTG showed that the recombinant protein could exist simultaneously in the inclusion body and soluble form of E. coli. The molecular weights were 45.61kDa 47.59kDa and 73kDa. respectively. The concentration of recombinant protein pET32a-753: 0.46 mg / ml, recombinant protein pET32a-807: 0.57 mg / ml, recombinant protein pET32a-1500: 0.51mg / ml were determined by using Ni ion chromatography column labeled with His. The results showed that the recombinant protein pET32a-753: 0.46 mg / ml, pET32a-807: 0.57 mg / ml, and pET32a-1500: 0.51 mg / ml, respectively. The serum titers of immunized mice were detected by Elisa according to the immune procedure. The spleen cells of immunized mice were fused with SP2/0 cells. The supernatant titers were detected by ELISA method after screening by HAT conditioned medium. According to the OD450nm value, the cell lines with high expression were screened. After the expansion of the positive cells selected by limited dilution, ascites were prepared by injecting mice intraperitoneally. The reactivity and classification of the prepared ascites were identified. The results showed that 227 positive monoclonal cell lines were selected from the supernatant of the culture medium for the first time, and 15 positive strains were screened after repeated tests. According to the OD450nm value, 5 of them were selected with higher OD450nm value. They were named as: 807-1D3807-1D6807-3E5A1500-3D21500-4G2. Finally, a stable positive strain: 807-1D6 was selected and its antibody class was IgG1. The recombinant KHV protein pET32a-807 was identified by Western-blot.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S941.41

【参考文献】